Circulating microRNAs in association with pancreatic cancer risk within 5 years.

Early detection is critical for improving pancreatic cancer prognosis. Our study aims to identify circulating microRNAs (miRNAs) associated with pancreatic cancer risk. The two-stage study used plasma samples collected ≤5 years prior to cancer diagnosis, from case-control studies nested in five prospective cohort studies. The discovery stage included 185 case-control pairs from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Replication stage samples comprised 277 pairs from Shanghai Women's Health Study/Shanghai Men's Health Study, Southern Community Cohort Study, and Multiethnic Cohort Study. Seven hundred and ninety-eight miRNAs were measured using the NanoString nCounter Analysis System. Odds ratios (OR) and 95% confidence intervals (CI) for per 10% change in miRNAs in association with pancreatic cancer risk were derived from conditional logistic regression analysis in discovery and replication studies, separately, and then meta-analyzed. Stratified analysis was conducted by age at diagnosis (<65/≥65 years) and time interval between sample collection and diagnosis (≤2/>2 years). In the discovery stage, 120 risk associated miRNAs were identified at p < .05. Three were validated in the replication stage: hsa-miR-199a-3p/hsa-miR-199b-3p, hsa-miR-767-5p, and hsa-miR-191-5p, with respective ORs (95% CI) being 0.89 (0.84-0.95), 1.08 (1.02-1.13), and 0.90 (0.85-0.95). Five additional miRNAs, hsa-miR-640, hsa-miR-874-5p, hsa-miR-1299, hsa-miR-22-3p, and hsa-miR-449b-5p, were validated among patients diagnosed at ≥65 years, with OR (95% CI) of 1.23 (1.09-1.39), 1.33 (1.16-1.52), 1.25 (1.09-1.43), 1.28 (1.12-1.46), 0.76 (0.65-0.89), and 1.22 (1.07-1.39), respectively. The miRNA targets were enriched in pancreatic carcinogenesis/progression-related pathways. Our study suggests that circulating miRNAs may identify individuals at high risk for pancreatic cancer ≤5 years prior to diagnosis, indicating its potential utility in cancer screening and surveillance.

Drug-target Mendelian randomization revealed a significant association of genetically proxied metformin effects with increased prostate cancer risk.

The association between metformin use and risk of prostate cancer remains controversial, while data from randomized trials is lacking. We aim to evaluate the association of genetically proxied metformin effects with prostate cancer risk using a drug-target Mendelian randomization (MR) approach. Summary statistics for prostate cancer were obtained from the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome Consortium (79,148 cases and 61,106 controls). Cis-expression quantitative trait loci (cis-eQTL) variants in the gene targets of metformin were identified in the GTEx project and eQTLGen consortium. We also obtained male-specific genome-wide association study data for type 2 diabetes, body mass index (BMI), total testosterone, bioavailable testosterone, estradiol, and sex hormone binding globulin for mediation analysis. Inverse-variance weighted (IVW) regression, weighted median, MR-Egger regression, and MR-PRESSO were performed in the main MR analysis. Multivariable MR was used to identify potential mediators and genetic colocalization analysis was performed to assess any shared genetic basis between two traits of interest. We found that genetically proxied metformin effects (1-SD HbA1c reduction, equivalent to 6.75 mmol/mol) were associated with higher risk of prostate cancer (odds ratioIVW [ORIVW]: 1.55, 95% confidence interval, CI: 1.23-1.96, p = 3.0 × 10-3). Two metformin targets, mitochondrial complex I (ORIVW: 1.48, 95% CI: 1.07-2.03, p = 0.016) and gamma-secretase complex (ORIVW: 2.58, 95%CI :1.47-4.55, p = 0.001), showed robust associations with prostate cancer risk, and their effects were partly mediated through BMI (16.4%) and total testosterone levels (34.3%), respectively. These results were further supported by colocalization analysis that expressions of NDUFA13 and BMI, APH1A, and total testosterone may be influenced by shared genetic factors, respectively. In summary, our study indicated that genetically proxied metformin effects may be associated with an increased risk of prostate cancer. Repurposing metformin for prostate cancer prevention in general populations is not supported by our findings.

One-year efficacy of myopia control by the defocus distributed multipoint lens: a multicentric randomised controlled trial.

AIMS: To report the 1-year results of the efficacy of a defocus distributed multipoint (DDM) lens in controlling myopia progression in a multicentre, randomised controlled trial. METHODS: Overall, 168 children aged 6-13 years were recruited and randomly assigned to wear a DDM lens (n=84) or single-vision (SV) lens (n=84) in three centres. Cycloplegic autorefraction (spherical equivalent refraction (SER)) and axial length (AL) were measured. Linear mixed model analysis was performed to compare between-group SER and AL changes. Logistic regression analysis was used to analyse the between-group difference in rapid myopia progression (SER increase≥0.75 D per year or AL growth≥0.40 mm per year). RESULTS: After 1 year, mean changes in SER were significantly lower in the DDM group (-0.47±0.37 D) than in the SV group (-0.71±0.42 D) (p<0.001). Similarly, mean changes in AL were significantly lower in the DDM group (0.21±0.17 mm) than in the SV group (0.34±0.16 mm) (p<0.001). After adjusting for age, sex, daily wearing time and parental myopia, rapid myopia progression risk was higher in the SV group than in the DDM group (OR=3.51, 95% CI: 1.77 to 6.99), especially for children who wore a lens for >12 hours per day, boys and younger children (6-9 years) with ORs (95% CIs) of 10.82 (3.22 to 36.37), 5.34 (1.93 to 14.78) and 8.73 (2.6 to 29.33), respectively. CONCLUSIONS: After 1 year, DDM lenses effectively retarded myopia progression in children. Longer daily wearing time of DDM lens improved the efficacy of myopia control. Future long-term studies are needed for validation. TRIAL REGISTRATION NUMBER: NCT05340699.

An expression-directed linear mixed model discovering low-effect genetic variants.

Detecting genetic variants with low-effect sizes using a moderate sample size is difficult, hindering downstream efforts to learn pathology and estimating heritability. In this work, by utilizing informative weights learned from training genetically predicted gene expression models, we formed an alternative approach to estimate the polygenic term in a linear mixed model. Our linear mixed model estimates the genetic background by incorporating their relevance to gene expression. Our protocol, expression-directed linear mixed model, enables the discovery of subtle signals of low-effect variants using moderate sample size. By applying expression-directed linear mixed model to cohorts of around 5,000 individuals with either binary (WTCCC) or quantitative (NFBC1966) traits, we demonstrated its power gain at the low-effect end of the genetic etiology spectrum. In aggregate, the additional low-effect variants detected by expression-directed linear mixed model substantially improved estimation of missing heritability. Expression-directed linear mixed model moves precision medicine forward by accurately detecting the contribution of low-effect genetic variants to human diseases.

Large-Scale Cancer Genomic Analysis Reveals Significant Disparities Between Microsatellite Instability and Tumor Mutational Burden.

BACKGROUND: Microsatellite instability (MSI) and tumor mutational burden (TMB) are predictive biomarkers for pan-cancer immunotherapy. The interrelationship between MSI-high (MSI-H) and TMB-high (TMB-H) in human cancers and their predictive value for immunotherapy in lung cancer remain unclear. METHODS: We analyzed somatic mutation data from the Genomics Evidence Neoplasia Information Exchange (n = 46,320) to determine the relationship between MSI-H and TMB-H in human cancers using adjusted multivariate regression models. Patient survival was examined using the Cox proportional hazards model. The association between MSI and genetic mutations was assessed. RESULTS: Patients (31-89 %) with MSI-H had TMB-low phenotypes across 22 cancer types. Colorectal and stomach cancers showed the strongest association between TMB and MSI. TMB-H patients with lung cancer who received immunotherapy exhibited significantly higher overall survival (hazard ratio of 0.61, 95% confidence interval: 0.44-0.86) and progression-free survival (0.65, 0.47-0.91) compared to the TMB-low group; no significant benefit was observed in the MSI-H group. Patients with TMB and MSI phenotypes showed further improvement in overall and progression-free survival. We identified several mutated genes associated with MSI-H phenotypes, including known mismatch repair genes and novel mutated genes, such as ARID1A and ARID1B. CONCLUSIONS: Our results demonstrate that TMB-H and/or a combination of MSI-H can serve as biomarkers for immunotherapies in lung cancer. IMPACT: These findings suggest that distinct or combined biomarkers should be considered for immunotherapy in human cancers because notable discrepancies exist between MSI-H and TMB-H across different cancer types.

Novel breast cancer susceptibility loci under linkage peaks identified in African ancestry consortia.

BACKGROUND: Expansion of genome-wide association studies across population groups is needed to improve our understanding of shared and unique genetic contributions to breast cancer. We performed association and replication studies guided by a priori linkage findings from African ancestry (AA) relative pairs. METHODS: We performed fixed-effect inverse-variance weighted meta-analysis under three significant AA breast cancer linkage peaks (3q26-27, 12q22-23, and 16q21-22) in 9241 AA cases and 10 193 AA controls. We examined associations with overall breast cancer as well as estrogen receptor (ER)-positive and negative subtypes (193,132 SNPs). We replicated associations in the African-ancestry Breast Cancer Genetic Consortium (AABCG). RESULTS: In AA women, we identified two associations on chr12q for overall breast cancer (rs1420647, OR = 1.15, p = 2.50×10-6; rs12322371, OR = 1.14, p = 3.15×10-6), and one for ER-negative breast cancer (rs77006600, OR = 1.67, p = 3.51×10-6). On chr3, we identified two associations with ER-negative disease (rs184090918, OR = 3.70, p = 1.23×10-5; rs76959804, OR = 3.57, p = 1.77×10-5) and on chr16q we identified an association with ER-negative disease (rs34147411, OR = 1.62, p = 8.82×10-6). In the replication study, the chr3 associations were significant and effect sizes were larger (rs184090918, OR: 6.66, 95% CI: 1.43, 31.01; rs76959804, OR: 5.24, 95% CI: 1.70, 16.16). CONCLUSION: The two chr3 SNPs are upstream to open chromatin ENSR00000710716, a regulatory feature that is actively regulated in mammary tissues, providing evidence that variants in this chr3 region may have a regulatory role in our target organ. Our study provides support for breast cancer variant discovery using prioritization based on linkage evidence.

A large-scale microRNA transcriptome-wide association study identifies two susceptibility microRNAs, miR-1307-5p and miR-192-3p, for colorectal cancer risk.

Transcriptome-wide association studies (TWAS) have identified many putative susceptibility genes for colorectal cancer (CRC) risk. However, susceptibility miRNAs, critical dysregulators of gene expression, remain unexplored. We genotyped DNA samples from 313 CRC East Asian patients and performed small RNA sequencing in their normal colon tissues distant from tumors to build genetic models for predicting miRNA expression. We applied these models and data from genome-wide association studies (GWAS) including 23 942 cases and 217 267 controls of East Asian ancestry to investigate associations of predicted miRNA expression with CRC risk. Perturbation experiments separately by promoting and inhibiting miRNAs expressions and further in vitro assays in both SW480 and HCT116 cells were conducted. At a Bonferroni-corrected threshold of P < 4.5 × 10-4, we identified two putative susceptibility miRNAs, miR-1307-5p and miR-192-3p, located in regions more than 500 kb away from any GWAS-identified risk variants in CRC. We observed that a high predicted expression of miR-1307-5p was associated with increased CRC risk, while a low predicted expression of miR-192-3p was associated with increased CRC risk. Our experimental results further provide strong evidence of their susceptible roles by showing that miR-1307-5p and miR-192-3p play a regulatory role, respectively, in promoting and inhibiting CRC cell proliferation, migration, and invasion, which was consistently observed in both SW480 and HCT116 cells. Our study provides additional insights into the biological mechanisms underlying CRC development.

Novel insights into genetic susceptibility for colorectal cancer from transcriptome-wide association and functional investigation.

BACKGROUND: Transcriptome-wide association studies have been successful in identifying candidate susceptibility genes for colorectal cancer (CRC). To strengthen susceptibility gene discovery, we conducted a large transcriptome-wide association study and an alternative splicing transcriptome-wide association study in CRC using improved genetic prediction models and performed in-depth functional investigations. METHODS: We analyzed RNA-sequencing data from normal colon tissues and genotype data from 423 European descendants to build genetic prediction models of gene expression and alternative splicing and evaluated model performance using independent RNA-sequencing data from normal colon tissues of the Genotype-Tissue Expression Project. We applied the verified models to genome-wide association studies (GWAS) summary statistics among 58 131 CRC cases and 67 347 controls of European ancestry to evaluate associations of genetically predicted gene expression and alternative splicing with CRC risk. We performed in vitro functional assays for 3 selected genes in multiple CRC cell lines. RESULTS: We identified 57 putative CRC susceptibility genes, which included the 48 genes from transcriptome-wide association studies and 15 genes from splicing transcriptome-wide association studies, at a Bonferroni-corrected P value less than .05. Of these, 16 genes were not previously implicated in CRC susceptibility, including a gene PDE7B (6q23.3) at locus previously not reported by CRC GWAS. Gene knockdown experiments confirmed the oncogenic roles for 2 unreported genes, TRPS1 and METRNL, and a recently reported gene, C14orf166. CONCLUSION: This study discovered new putative susceptibility genes of CRC and provided novel insights into the biological mechanisms underlying CRC development.

Regulation of secondary metabolites in the endophytic fungus Penicillium sp. KMU18029 by the chemical epigenetic modifier 5-azacitidine.

The chemical epigenetic modifier 5-azacitidine (5-Aza C), a DNA methyltransferase inhibitor, was used to manipulate the endophytic fungus Penicillium sp. KMU18029. From its rice fermentation extract, a new polyketone compound (3S,4R)-3,4,8-trihydroxy-6-methyl-3,4-dihydronaphthalen-1(2H)-one (1), along with 13 known compounds, 3,4,8-trihydroxy-6-(hydroxymethyl)-3,4-dihydronaphthalen-1(2H)-one (2), decaturin B (3), 15-hydroxydecaturin A (4), oxalicine A (5), pileotin A (6), pyrandecarurin A (7), decaturenol A (8), decaturenoid (9), penisarins A (10), oxaline (11), (4E,8E)-N-D-2'-hydroxyocta-decanoyl-1-O-ß-D-glycopy-ranosyl-9-methyl-4,8-sphingadienine (12), ergosterol (13) and stigma-5-en-3-O-ß-glucoside (14), were separated. Among the known compounds, 2, 7, 12 and 14 were not found in our previous research on this strain. The structure of the new compound was identified by spectroscopic techniques such as HR-ESIMS, 1D NMR, 2D NMR and CD. Furthermore, all the isolated compounds were tested for their antimicrobial activities, and only compounds 1, 2 and 11 showed weak activities against S. aureus, with MICs of 128 µg/mL.

High intake of cruciferous vegetables reduces the risk of gastrointestinal cancers: results from observational studies.

The relationship between cruciferous vegetables (CV) and the risk of gastrointestinal (GI) cancers has been extensively investigated. However, epidemiologic investigations have produced inconsistent results. This meta-analysis investigated the association between CV intake and the risk of GI cancers. Due to the heterogeneity, fixed- or random-effects models were used for the analyses. The final analysis included 81 articles covering 89 studies. In comparison to the lowest consumption categories, the highest consumption categories of CV were associated with a lower risk for all GI cancers [rate ratio (RR): 0.81, 95% confidence interval (95% CI) 0.76-0.87]. Compared to a CV intake of 75 g/day, subjects with CV intake <75 g/day experienced a 7% reduction in risk (RR: 0.93; 95% CI: 0.84-0.96) for each 50 g increase in consumption. A negative correlation was identified between CV intake and the risk of esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, and colorectal cancer (CRC), but not gallbladder cancer (RR: 0.70; 95% CI: 0.38-1.27). High intake of broccoli and cabbage was associated with a decreased risk of gastric cancer (RR: 0.64; 95% CI: 0.47-0.87) and gallbladder cancer (RR: 0.46; 95% CI: 0.29-0.75). These results confirm the association between high intake of CV with a reduced risk of GI cancers.

cLD: Rare-variant linkage disequilibrium between genomic regions identifies novel genomic interactions.

Linkage disequilibrium (LD) is a fundamental concept in genetics; critical for studying genetic associations and molecular evolution. However, LD measurements are only reliable for common genetic variants, leaving low-frequency variants unanalyzed. In this work, we introduce cumulative LD (cLD), a stable statistic that captures the rare-variant LD between genetic regions, which reflects more biological interactions between variants, in addition to lack of recombination. We derived the theoretical variance of cLD using delta methods to demonstrate its higher stability than LD for rare variants. This property is also verified by bootstrapped simulations using real data. In application, we find cLD reveals an increased genetic association between genes in 3D chromatin interactions, a phenomenon recently reported negatively by calculating standard LD between common variants. Additionally, we show that cLD is higher between gene pairs reported in interaction databases, identifies unreported protein-protein interactions, and reveals interacting genes distinguishing case/control samples in association studies.

Large-scale alternative polyadenylation (APA)-wide association studies to identify putative susceptibility genes in human common cancers.

Alternative polyadenylation (APA) modulates mRNA processing in the 3' untranslated regions (3'UTR), which affect mRNA stability and translation efficiency. Here, we build genetic models to predict APA levels in multiple tissues using sequencing data of 1,337 samples from the Genotype-Tissue Expression, and apply these models to assess associations between genetically predicted APA levels and cancer risk with data from large genome-wide association studies of six common cancers, including breast, ovary, prostate, colorectum, lung, and pancreas among European-ancestry populations. At a Bonferroni-corrected P □<□0.05, we identify 58 risk genes, including seven in newly identified loci. Using luciferase reporter assays, we demonstrate that risk alleles of 3'UTR variants, rs324015 ( STAT6 ), rs2280503 ( DIP2B ), rs1128450 ( FBXO38 ) and rs145220637 ( LDAH ), could significantly increase post-transcriptional activities of their target genes compared to reference alleles. Further gene knockdown experiments confirm their oncogenic roles. Our study provides additional insight into the genetic susceptibility of these common cancers.

An exploratory study of knowledge, attitudes, and practices toward HPV associated anal cancer among Pakistani population.

Background: Anal cancer, mainly attributed to human papillomavirus (HPV) infection, is rising in prevalence among the general population in Pakistan. This study aimed to examine the knowledge, attitudes, and practices (KAP) towards anal cancer screening and HPV of the general population in Pakistan. Method: We surveyed anal cancer KAP using social media and snowball sampling from December 2022 to May 2023. The questionnaire had 16 knowledge, 12 attitudes, 6 practice questions, and socio-demographic variables. We applied validity criteria for inclusion and exclusion and used cutoffs ≥50% for each KAP category. We analyzed data in R with Guttman's λ2 for reliability, did univariate and bivariate analysis, and reported frequencies, percentages, p-values, coefficients, odds ratios, and 95% confidence intervals. Results: We surveyed 1620 people and discovered low awareness of HPV and anal cancer causes prevention, and screening (11%-24%), high stigma and embarrassment for screening (54%-70%), strong moral beliefs (89%), condom nonuse (91%), and low engagement in health services and programs (9.1%-14%). Knowledge (75.23%, OR = 1.0984, p = 0.05) was shaped by socio-demographic factors, attitude, and practice, with higher education enhancing knowledge (OR = 1.0984, p = 0.05). Attitude (78.45%, OR = 6.6052, p< 0.001) was influenced by socio-demographic factors, practice, and knowledge as well. Younger females, single, unemployed, students, living with more family members, earning more income, and residing in Islamabad had a more positive attitude (ORs from 1.0115 to 6.6052, p< 0.05), while religion did not affect attitude (p = 0.51). Practice (9.16%, OR = 0.1820, p< 0.001) was determined by socio-demographic factors, knowledge, and attitude. Older males, employed teachers, living with more family members, earning less income, and residing in Islamabad had better practice (ORs from 0.1323 to 3.8431, p< 0.05), but marital status and religion did not influence practice (p > 0.05). Conclusion: Pakistani young adults need more education, awareness, health services, and programs on HPV and anal cancer, as they have low awareness, high stigma, and socio-cultural challenges. In addition, it is recommended for more research and policy initiatives are needed to address socio-cultural factors and increase anal Pap to overcome anal cancer.

Enhancing Disease Risk Gene Discovery by Integrating Transcription Factor-Linked Trans-located Variants into Transcriptome-Wide Association Analyses.

Transcriptome-wide association studies (TWAS) have been successful in identifying putative disease susceptibility genes by integrating gene expression predictions with genome-wide association studies (GWAS) data. However, current TWAS models only consider cis-located variants to predict gene expression. Here, we introduce transTF-TWAS, which includes transcription factor (TF)-linked trans-located variants for model building. Using data from the Genotype-Tissue Expression project, we predict alternative splicing and gene expression and applied these models to large GWAS datasets for breast, prostate, and lung cancers. Our analysis revealed 887 putative cancer susceptibility genes, including 465 in regions not yet reported by previous GWAS and 137 in known GWAS loci but not yet reported previously, at Bonferroni-corrected P < 0.05. We demonstrate that transTF-TWAS surpasses other approaches in both building gene prediction models and identifying disease-associated genes. These results have shed new light on several genetically driven key regulators and their associated regulatory networks underlying disease susceptibility.

Integrating genomics and proteomics data to identify candidate plasma biomarkers for lung cancer risk among European descendants.

BACKGROUND: Plasma proteins are potential biomarkers for complex diseases. We aimed to identify plasma protein biomarkers for lung cancer. METHODS: We investigated genetically predicted plasma levels of 1130 proteins in association with lung cancer risk among 29,266 cases and 56,450 controls of European descent. For proteins significantly associated with lung cancer risk, we evaluated associations of genetically predicted expression of their coding genes with the risk of lung cancer. RESULTS: Nine proteins were identified with genetically predicted plasma levels significantly associated with overall lung cancer risk at a false discovery rate (FDR) of <0.05. Proteins C2, MICA, AIF1, and CTSH were associated with increased lung cancer risk, while proteins SFTPB, HLA-DQA2, MICB, NRP1, and GMFG were associated with decreased lung cancer risk. Stratified analyses by histological types revealed the cross-subtype consistency of these nine associations and identified an additional protein, ICAM5, significantly associated with lung adenocarcinoma risk (FDR < 0.05). Coding genes of NRP1 and ICAM5 proteins are located at two loci that have never been reported by previous GWAS. Genetically predicted blood levels of genes C2, AIF1, and CTSH were associated with lung cancer risk, in directions consistent with those shown in protein-level analyses. CONCLUSION: Identification of novel plasma protein biomarkers provided new insights into the biology of lung cancer.

An updated systemic review and meta-analysis on human papillomavirus in breast carcinogenesis.

Background: Breast Cancer (BC) stands out as the widely prevalent malignancy among all the types of cancer affecting women worldwide. There is significant evidence that the pathogenicity of BC may be altered by Human Papillomavirus (HPV) infection; however, conclusive data are not yet available. Methods: By searching five databases, including EMBASE, IBECS, PubMed, Scopus, Science Direct, Google Scholar, and Web of Science, a thorough systematic analysis was conducted on the prevalence of HPV in BC patients from 1990 to June 30, 2022. After applying extensive eligibility criteria, we selected 74 publications for further analysis based on the prevalence of HPV infections in breast tissues. All of the data were analyzed using a random-effects meta-analysis, Cochran Q test and I2 statistic were used to calculate the heterogeneity of the prevalence among these studies using subgroup analysis. Variations in the HPV prevalence estimates in different subgroups were evaluated by subgroup meta-analysis. Results: In total, 3156 studies were initially screened, resulting in 93 full-text studies reviewed, with 74 meeting inclusion criteria. Among a total of 7156 BC biopsies, the pool prevalence of HPV was 25.6% (95% CI= 0.24-0.33, τ2 = 0.0369 with significant heterogeneity between estimates (I 2 = 97% and p< 0.01). Consequently, 45 studies with available controls were further studied, and the prevalence of HPV in case-control studies was 26.2% with overall odds 5.55 (95% CI= 3.67-8.41, I 2 = 38%, τ2 = 1.4878, p< 0.01). Further subgroup analysis of HPV revealed HPV-16 had a maximum prevalence of 9.6% (95% CI= 3.06-11.86, I 2 = 0%, τ2 = 0.6111, p< 0.01). Among different geographical regions, Europe reported the maximum prevalence of HPV, i.e., 39.2% (95% CI=1.29-7.91, I 2 = 18%, τ2 = 1.2911, p< 0.01). Overall distribution showed HPV-18 was a frequent HPV subtype reported in Australia. Conclusion: Current study provides a global estimate of HPV prevalence in BC patients and demonstrates a significant association between this virus and BC etiology. Nevertheless, we recommend further investigation into the underlying mechanism is essential to validate this hypothesis.

Frame rate effects and their compensation on super-resolution microvessel imaging using ultrasound localization microscopy.

Ultrasound localization microscopy (ULM) breaks the diffraction limit and allows imaging microvasculature at micrometric resolution while preserving the penetration depth. Frame rate plays an important role for high-quality ULM imaging, but there is still a lack of review and investigation of the frame rate effects on ULM. This work aims to clarify how frame rate influences the performance of ULM, including the effects of microbubble detection, localization and tracking. The performance of ULM was evaluated using an in vivo rat brain dataset (15.6 MHz, 3 tilted plane waves (-5°, 0°, +5°), at a compounded frame rate of 1000 Hz) with different frame rates. Quantification methods, including Fourier ring correlation and saturation parameter, were applied to analyze the spatial resolution and reconstruction efficiency, respectively. In addition, effects on each crucial step in ULM processing were further analyzed. Results showed that when frame rates dropped from 1000 Hz to 250 Hz, the spatial resolution deteriorated from 9.9 µm to 15.0 µm. Applying a velocity constraint was able to improve the ULM performance, but inappropriate constraint may artificially result in high apparent resolution. For the dataset, compared with the results of 1000 Hz frame rate, the velocity was underestimated at 100 Hz with 47.18% difference and the saturation was reduced from 55.00% at 1000 Hz to 43.34% at 100 Hz. Analysis showed that inadequate frame rate generated unreliable microbubble detection, localization and tracking as well as incomplete track reconstruction, resulting in the deterioration in spatial resolution, the underestimation in velocity measurement and the decrease in saturation. Finally, a guidance of determining the frame rate requirement was discussed by considering the required spatial sampling points based on vessel morphology, clutter filtering method, tracking algorithm and acquisition time, which provides indications for future clinical application of ULM method.